Fig 1.
Pedigrees with novel mutations.
The segregation analysis of 22 pedigrees showed 26 rare potentially pathogenic novel variants along with 8 previously reported mutations in 17 IRD associated genes. These include 9 homozygous, 21 compound heterozygous, two dominant acting heterozygous, and two X-linked variants. Pedigrees A-I are Mexican, J-M are Pakistani and N-V are European American. The asterisk indicates the availability of whole-genome sequencing data.
Table 1.
Pedigrees with novel potentially causative variants in known IRD genes.
Fig 2.
Pedigrees with previously reported mutations.
The segregation analysis revealed 35 previously reported mutations that were identified in 25 pedigrees. There were seven frameshift, 17 missense, seven premature stop codon mutations, and four splice site altering changes. Of these ten are homozygous, six dominant heterozygous, two X-linked, and 17 compound heterozygous mutations found in these pedigrees. Pedigrees A-H are Mexican, I-K are Pakistani and L-V are European American, W-X are Ashkenazi Jewish and Y is Indian. The asterisk indicates the availability of whole-genome sequencing data.
Table 2.
Pedigrees with previously reported mutations in IRD genes.
Fig 3.
Pedigrees with copy number variations (CNVs).
Five unique structural variants were identified in EYS, LCA5, CERKL, PRPH2, and CNGB3 in five different pedigrees, one with dominant macular degeneration and four with recessive retinal degeneration. (A1) 1.6Mb homozygous deletion Chr6: g.65,994,849_67,582,755del is segregating with recessive retinal degeneration in a Mexican pedigree RF.V196.0210. (A2) The schematic diagram depicting a 1.6Mb homozygous deletion Chr6: g.65,994,849_67,582,755del encompassing the exons 1 to 12 and about 1.6Mb of 5’-untranslated region of the EYS gene. (A3) PCR amplification of EYS exons 1, 6, and 11 detected the presence of expected size product in unaffected individuals (I:1, I:2, II:3) whereas the presence of PCR product was not observed in two affected individuals (II:1 and II:2). (A4) Amplification with primers flanking the deleted region followed by sequencing revealed the junction point in individual II:1 due to the 1.6Mb deletion. Examination of the sequence flanking the junction point detected paralogous repeat sequences on both sides of the deleted region (blue and yellow boxes). (B1) In a consanguineous Pakistani pedigree RF.277.0113, a 110Kb homozygous deletion in LCA5 (chr6: g.80,205,052_80,315,592del) segregated with the phenotype. (B2) The novel 110Kb homozygous deletion includes 1 to 4 exons of LCA5. (B3) PCR amplification showed the absence of exon 1 to 4 of LCA5 in both affected individuals while exon 5 is present in all family members. (B4) Amplification with primers flanking the deletion resulted in the generation of the fragment with deletion. Sequencing this PCR product revealed the presence of paralogous repeat sequences flanking the junction point in affected individuals. Sequence marked with yellow and blue rectangles represent the paralogous sequence on both sides of the deleted region. (C1) A previously reported heterozygous stop mutation CERKL p.Arg257* and a novel heterozygous large 22.8 Kb deletion (Chr2: g.182,456,422_182,479,267del) on chromosome 2, which includes exon 2 of CERKL are observed in trans configuration in the proband of RF.T.8.11. (C2) The schematic diagram shows the 22.8Kb deletion which includes ~10.3Kb of intron 1 and exon 2 (243bp) and 12.3Kb of intron 2 of CERKL gene. (C3) The WGS reads mapped to the deleted region showed decrease in read depth. (C4) Electropherogram showing the sequence of junction fragment generated by amplification with primers flanking the deletion revealed the specific boundaries of the deletion that includes exon 2 of CERKL. (D1) The segregation analysis revealed a heterozygous 33Kb deletion on chromosome 6 (Chr6: g.42,643,442_42,676,411del) segregating with the disease. (D2) A cartoon depicting the deleted region which includes two different genes: exons 39 and 40 of UBR2 and exons 2 and 3 of PRPH2 present in opposite orientation (D3) Analysis of 7 affected and 15 unaffected members using qPCR confirmed the presence of the heterozygous deletion on chromosome 6 in affected members and not in unaffected relatives. (E1) A set of compound heterozygous deletions including a novel 7Kb deletion (Chr8: g.87,616,103_87,623,431del) and a previously known 7bp deletion p.Arg274Valfs*13 in CNGB3 gene were observed in RF.M.0592 pedigree with a single affected individual. (E2) The novel 7Kb heterozygous deletion (Chr8: g.87,616,103_87,623,431del) (Pink rectangle) includes coding exon 15 of CNGB3. (E3) qPCR analysis confirmed the presence of the heterozygous deletion of CNGB3 exon 15 in II:1, which was inherited from the mother (I:2). The asterisk indicates the availability of whole-genome sequencing data.
Table 3.
Pedigrees with copy number variations in IRD genes.
Fig 4.
Segregation analysis of pedigrees with atypical genotypes.
A-D: Mexican pedigrees with atypical mutations. A. RF.VI13.0707 pedigree with a de-novo mutation in IMPDH1; B. RF.VI104.0514 pedigree with a homozygous nonsense mutation in C2orf71 was detected in generation II while a previously reported homozygous splice site mutation in the CLN3 gene was observed in proband in the IV generation demonstrating the involvement of two different genes in IRD pathology in different generations. C. RF.VI157.0216 pedigree with mutations in genes OPN1SW and TOPORS associated with dominant color blindness and retinitis pigmentosa; D. RF.VI111.0514 pedigree with novel heterozygous causative mutation in PRPF8 and a known mutation in PRPF31, each sufficient to cause dominant IRD, were observed in monozygotic affected twins; E. RF.197.0113 consanguineous pedigree from Pakistan with a previously known dominant acting mutation in PRPF3 segregated in a pseudo-recessive pattern; F. RF.M.1111 an European American pedigrees with causative variants in more than one known IRD genes were observed to segregate with disease. A homozygous mutation in PDE6G and a hemizygous mutation in OPN1LW were observed in Pedigree RF.M.1111. G. RF.K.0216 Indian pedigree with a heterozygous PRPH2 mutation that is sufficient to cause retinal dystrophy and an additional mutation in ROM1 that can lead to digenic RP along with the PRPH2 variant. The asterisk indicates the availability of whole-genome sequencing data.
Table 4.
Pedigrees with atypical genotypes in IRD genes.
Fig 5.
Haplotype of parents and the proband constructed with variants flanking the de-novo variant detected in the IMPDH1 gene in RF.VI13.0707 pedigree (Fig 4A).
Fig 6.
Summary of findings and population distribution of novel mutations detected in Mexican, Pakistani and European American pedigrees.
Table 5.
Summary of findings and population distribution of mutations detected in three major cohorts analyzed.
Fig 7.
Distribution of IRD genes detected in 61 pedigrees.